Process for enzyme immobilization on a fluorocarbon surface

ABSTRACT

The fluorocarbon surface of a solid or liquid support is activated with a highly fluorinated isocyanate-modified ligand or with a reactive poly(fluoroalkyl) sugar reagent containing a polyhydroxy sugar to which are attached a plurality of fluoroalkyl anchor groups, a reactive group and optionally a spacer. The activated support has application in separation of biomolecules, immobilization of biomolecules, heterogeneous diagnostic assays, and biosensors. An enzyme or other biomolecule is immobilized by contacting the activated support surface with the enzyme in the presence of a surfactant. The surfactant is preferably a neutral surfactant such as a fluoroalkyl-polyoxyethylene.

This application is a continuation of application Ser. No. 07/586,183,filed Sep. 21, 1990, now abandoned.

TECHNICAL FIELD

This invention relates to a novel group of poly(flouroalkyl) sugarreagents, a method for their use for the modification of the surface ofsolid or liquid supports, and supports used for application in theseparation of biomolecules, enzyme immobilization, heterogeneousdiagnostic assays, and biosensors.

BACKGROUND ART

Numerous methods have been developed for the immobilization of proteinsand other biomolecules onto solid or liquid supports. A description ofthese methods can be found in general reviews such as that given byMosbach, 1976, Methods in Enzymology, Vol. 44; Weetall, 1975,Immobilized Enzymes, Antigens, Antibodies, and Peptides; or Kennedy etal., 1983, Solid Phase Biochemistry, Analytical and Synthetic Aspects,Scouten, ed., pp. 253-391. The most commonly used methods are adsorptionor covalent binding to the support.

Adsorption is the oldest and simplest method for protein immobilization.To effect immobilization, a solution of the protein is contacted with asupport material such as alumina, carbon, an ion-exchange resin,cellulose, glass or a ceramic. Although the immobilization procedure maybe simple, the interactions involved in the adsorption process arecomplex and include charge-charge, van der Waals and hydrophobicinteractions, and hydrogen bonding. The adsorption method has theadvantages of low cost, extreme simplicity, mild immobilizationconditions and the ability to regenerate the support. The mainlimitation of this method is the relatively weak interaction between theprotein and the support, which may result in desorption of the proteinupon changes in pH and ionic strength. The often undefined nature ofthese interactions also can limit their use.

The most frequently used immobilization technique is the covalentbinding of the protein to chemically activated solid supports such asglass, synthetic polymers, and cross-linked polysaccharides. (Generally,this technique results in a protein which is immobilized in a morestable fashion than protein immobilized by adsorption.) An example ofthis method is the cyanogen bromide activation of polysaccharidesupports, e.g., agarose.

Although these traditional supports have been used in many applications,they suffer from some limitations. The polysaccharide supports arecompressible, which limits their application in column configurations athigh flow rates. These supports are also susceptible to microbialattack. Silica supports are not stable under alkaline conditions.Polymeric supports are also not chemically inert, and usually have aspecific gravity close to 1, which results in long settling times inbatch operations. Moreover, all of these supports exhibit varyingdegrees of nonspecific binding of unwanted proteins. The use of solidand liquid fluorocarbon supports overcome many of these limitations.Fluorocarbons are chemically inert and mechanically stable. The highspecific gravity of fluorocarbon supports results in rapid settling inbatch operations. However, it is difficult to activate fluorocarbonsupports for imobilization.

Fluorocarbon polymers have been used as supports to which biomoleculeshave been attached by adsorption [U.S. Pat. No. 3,843,443, issued toFishman on Oct. 22, 1974; WO 8603-840-A filed by Rijskuniv Groningen;Danielson and Siergiej, Biotechnol. Bioeng. 23, 1913-1917 (1981);Siergeiej, Dissertation Abstracts, Int. B., Volume 44, 153 (1983)].Because these methods rely on simple adsorption of the biomolecule ontothe support, the attachment is relatively weak. Consequently, some orall of the immobilized biomolecule is lost during use. In addition, asignificant loss of biological activity of the biomolecule results uponadsorption.

Busby et al. (U.S. Pat. No. 4,317,879, issued Mar. 2, 1982) disclose thecovalent attachment of the enzyme glucose oxidase to a fluorocarbonmembrane. The membrane was first etched with a sodium dispersion innaphthalene, followed by paraformaldehyde linking of the enzyme. Thismethod requires severe chemical conditions to activate the fluorocarbonsurface for covalent binding to the enzyme.

Hato et al., (U.S. Pat. No. 4,619,897, issued Oct. 23, 1986) disclosethe immobilization of enzymes onto a fluorine resin membrane which ismade hydrophilic on one side by penetration of a perfluoroalkyl surfaceactive agent to a prescribed depth. The asymmetrically functionalmembrane obtained is then treated with an enzyme and a cross-linkingagent such as glutaraldehyde to effect immobilization. In this approach,the fluorocarbon surface is not activated for covalent attachment of theenzyme. Rather, the enzyme is cross-linked within the pores of thewetted membrane. This approach is limited to porous fluorocarbonmembranes.

The use of perfluorocarbons polymer-based supports for enzymeimmobilization and affinity chromatography is described in U.S. Pat. No.4,885,250 issued Dec. 5, 1989. In this method the biomolecule is firstmodified by reaction with a perfluoroalkylating agent. Then, themodified protein is adsorbed onto the fluorocarbon support to effectimmobilization. This procedure works well for the immobilization of manybiomolecules, particularly immunoglobulins. However, substantial loss ofbiological activity results for some proteins because of the need to useorganic solvents (16% v/v) in the perfluoroalkylation reaction, thehydrophobic nature of the fluorocarbon support, and the need formultipoint modification of protein of obtain secure immobilization.Mulitpoint modification of the biomolecule is required because of themono(fluoroalkyl) reagents used. In addition, the mono(fluoroalkyl)reagents desorb from the support in the presence of high levels oforganic solvents, e.g., about 50% or greater.

Giaver, (U.S. Pat. No. 4,619,904, issued Oct. 28, 1986) describes theuse of fluorocarbon emulsions in agglutination immunoassays. Theemulsions were formed by adding a fluorinated polar molecule such aspentafluorobenzoyl chloride to a fluorocarbon liquid. The resultingemulsion was contacted with an aqueous solution of the protein. Again,mono(fluoroalkyl) anchor groups were used to immobilize the protein.

Lowe et al. in copending application Ser. No. 428,154, describe theattachment of biomolecules to fluorocarbon surfaces by means of apolymer such as poly(vinyl alcohol), which has been chemically modifiedto contain a significant number of perfluoroalkyl groups. Although thisapproach provides multiple fluoroalkyl anchor groups for secureattachment to the fluorocarbon surface, the number of anchor groups isdifficult to control and reproduce.

De Miguel et al., Chromatographia, Vol. 24, 849-853, 1987, describe thestrong retention of phenyl-D-glucopyranoside, modified with multiplefluorocarbon chains, on fluorocarbon bonded phases under reversed phaseconditions. The authors speculate that such strong retention may allowdynamic anchoring of biomolecules. No examples were provided. Thecompounds described cannot be used for immobilization because theycontain no reactive group to couple to the biomolecule. The majordifference between the phenol-D-glucopyranosides of De Miguel et al.,and the present invention is that their compounds do not contain aspacer arm and reactive group for covalent binding to the biomolecule.

SUMMARY OF THE INVENTION

This invention concerns (a) a process for immobilizing a biomolecule ona fluorocarbon surface comprising the steps of:

(1) activating the fluorocarbon surface, by, for example, contacting thesurface with a highly fluorinated isocyanate-modified ligand or bycontacting the fluorocarbon surface with a reactive poly(fluoroalkyl)sugar reagent containing a surface template to which are attached aplurality of fluoroalkyl anchor groups, an optional spacer and areactive group by causing the fluorocarbon surface to adsorb thereagent; then

(2) adding a solution of a biomolecule, in the presence of a surfactant,to the activated fluorocarbon surface to attach to the sugar reagent, toimmobilize the biomolecule on the fluorocarbon surface.

This invention further concerns (b) a process for immobilizing abiomolecule on a fluorocarbon surface comprising the steps of:

(1) activating the fluorocarbon surface by contacting the fluorocarbonsurface with an activator, for example a highly fluorinated isocyanateor a reactive poly(fluoroalkyl) sugar reagent containing a sugartemplate to which are attached a plurality of fluoroalkyl anchor groups,an optional spacer and a reactive group by causing the fluorocarbonsurface to adsorb the reagent; then

(2) adding a surfactant; then

(3) adding a solution of a biomolecule to the activated fluorocarbonsurface to attach to the sugar reagent to immobilize the biomolecule onthe fluorocarbon surface.

This invention also concerns (c) a process for immobilizing abiomolecule on a fluorocarbon surface comprising the steps of:

(1) activating the fluorocarbon surface by contacting the fluorocarbonsurface with an activator, for example a highly fluorinatedisocyanate-modified ligand or a reactive poly(fluoroalkyl) sugar reagentcontaining a sugar template to which are attached a plurality offluoroalkyl anchor groups, an optional spacer and a reactive group, inthe presence of a surfactant, by causing the fluorocarbon surface toadsorb the reagent; then

(2) adding a solution of a biomolecule to the activated fluorocarbonsurface to attach to the sugar reagent to immobilize the biomolecule onthe fluorocarbon surface.

DESCRIPTION OF INVENTION

The poly(fluoroalkyl) sugar reagents used in this invention contain asugar template to which are attached multiple fluoroalkyl anchor groups.The sugar can be a monosaccharide, such as glucose, mannose, galactose,gluconic acid, and glucoheptanoic acid, a disaccharide, such as maltoseand lactose, or any polyhydroxy compound with a well-defined number ofhydroxyl groups. These structures permit the attachment of multiplefluoroalkyl anchor groups. For example, glucose, gluconic acid andglucoheptanoic acid allow attachment of four, five and sixperfluoroalkyl anchor groups, respectively. The glucoheptanoic acidreagent is preferred because the six fluoroalkyl groups provide the mostsecure attachment of the reagent to the fluorocarbon surface.

The sugar reagents may be dissolved in an aqueous or mixed organicsolvent (see Example 1).

The sugar reagent group is a moiety containing at one end, a highlyfluorinated anchor group, such as perfluorobutyl, perfluorohexyl, orperfluorooctyl, capable of attaching to a fluorocarbon surface and atthe other end, a reactive group capable of covalent coupling to thebiomolecule. Examples of reactive groups include: carboxylic acid,amine, acylhydrazide, aldehyde, an active ester such asN-acyloxysuccinimide, acylimidazolide and epoxide. The anchor portionand the reactive group can be separated by a spacer group.Alternatively, reagents with a polyoxyethylene group can be used asneutral surfactants to prepare supports for size exclusionchromatography. Charged groups, such as quaternary ammonium ion andcarboxylic acids, are used to prepare ion-exchange supports useful inion-exchange chromatography and ion-exchange membranes.

Where the surface modifying compound is a highly fluorinated isocyanateanchor group, the anchor group is based on a compound having the formulaR_(F) --CH₂ CH₂ CH₂ --NCO, where R_(F) is a linear, branched orcarbocyclic perfluorinated radical containing 1-20 carbon atoms.

A general method of forming the solid or liquid support of thisinvention useful for bioseparations, enzyme immobilization, diagnosticassays, and biosensors, is to first activate the fluorocarbon support byadsorbing the poly(fluoroalkyl) sugar reagent onto the surface. Thepoly(fluoroalkyl) reagents are adsorbed much more strongly thanmono(fluoroalkyl) anchor reagents. The poly(fluoroalkyl) reagents adsorbonto fluorocarbon surfaces in the presence of high concentrations oforganic solvents, e.g., acetone, 50 to 90%, while mono(fluoroalkyl)reagents are desorbed from the fluorocarbon surface in the presence ofhigh levels of organic solvent, e.g., acetone, 50% or greater. To effectimmobilization, an aqueous solution of the biomolecule is added to theactivated fluorocarbon support.

A poly(fluoroalkyl) sugar reagent with a poly(oxyethylene) group, e.g.,reagent 38, a neutral fluorosurfactant, can be coimmobilized along withthe reactive poly(fluoroalkyl) sugar, to minimize nonspecific binding ofother proteins and to improve the retention of biological activity ofthe immobilizing reagent.

The surfactant also renders the surface more hydrophilic which improvesthe wettability of the support. Alternatively, a mono(fluoroalkyl)neutral fluorosurfactant, such as Zonyl® FSN fluorosurfactant, afluoroalkyl-polyoxyethylene surfactant, can be added to thepoly(fluoroalkyl) sugar reagent solution, the solution of thebiomolecule to be immobilized, or the support can be treated with thefluorosurfactant in a separate step either after or just preceding theimmobilization step.

The fluorocarbon surface can be a solid fluorocarbon polymer such aspoly(tetrafluoroethylene), a liquid fluorocarbon such asperfluorodecalin, or a nonfluorocarbon support that is coated with afluorocarbon interlayer.

The non-fluorocarbon core for preparing solid supports of this inventioninclude inorganic surfaces such as silica, magnetic particles andpolymers such as polystyrene, polypropylene and polyethylene. Byinterlayer is meant a layer of fluorocarbon coating located on thesurface of the non-fluorocarbon solid carrier core. The fluorocarboncompound can be a fluoropolymer, fluorosilane or other highlyfluorinated hydrocarbon chain. The interlayer may be applied to anon-fluorocarbon solid by spray coating or by chemical reactions. Asufficient amount of fluorocarbon surface must be present to secure thereagent and anchor the biomolecule.

The process of the present invention can be used in the preparation ofsupports for the separation of biomolecules, enzyme immobilization,heterogeneous diagnostic assays and biosensors.

The main advantages of the use of poly(fluoroalkyl) sugar reagents overthe mono(fluoroalkyl) reagents are: higher retention of biologicalactivity, higher immobilization efficiency, more secure attachment ofthe biomolecule, a more stable preactivated support, and a simplerimmobilization procedure. The supports described in this invention canbe used for various kinds of extra-corporeal depletion therapy, fornucleic acid hybridization assays, to capture DNA or RNA from mixturesand for various configurations of solid and liquid phase bioassays.

Below are the Experimental details for the synthesis ofpoly(fluoroalkyl) sugar reagents. The reagents will be numbered as shownbelow throughout the specification. ##STR1##

D-glucose 1, 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranoside 2,gluconolactone 18 and glucoheptanolactone 26 are commercially availableand were purchased from Aldrich Chemical Company.

2,3,4,6-Tetra-O-Acetyl-α-D-Glucopyranosyl Bromide 3

In a 250-mL round-bottom flask equipped with a magnetic stirring barwere placed 2 (19.577 g, 50 mmol), acetic anhydride (12.5 mL) andhydrogen bromide (75 mL, 30-32% solution by weight in glacial aceticacid) and allowed to stir at room temperature for 24 h. It was thenpoured into ice-cold dichloromethane (˜100 mL) in a separatory funnel,washed with ice-cold water (3×75 mL), saturated sodium bicarbonate. Theresidual after drying and removal of solvent was crystallized from amixture of diethyl ether and hexane to provide 3 (16.96 g, 82.7% yield).The compound 3 was identified by ¹ H NMR.

5'-Hexenyl 2,3,4,6-Tetra-O-Acetyl-β-D-Glucopyranoside 4

In a 500-mL round-bottom flask equipped with overhead mechanical stirrerand addition funnel were placed silver carbonate (11.03 g, 40 mmol),anhydrous calcium sulfate (drierite, ground with a mortar and pestlethen vacuum dried for an hour), 5-hexen-1-ol (4.8 mL, 40 mmol) andanhydrous dichloromethane (100 mL). Meanwhile, compound 3 (8.2 g, 20mmol) was dissolved in 75 mL of dichloromethane and added dropwise tothe above mixture over a 2-h period, and the solution stirred vigorouslyfor 48 h. The reaction mixture was filtered and the residue afterremoval of the solvent chromatographed (silica gel 300 g, 1:3 ethylacetate/hexane) to provide 4 (4.95 g, 57.5% yield).

¹ H NMR (300 MHz; CDCl₃ δ 1.43 (m, 2H, H-3), 1.583 (m, 2H, H-2'), 2.007,2.025, 2.037, 2.087 (4 s+m, 14H, 4 OAc, +H-4'), 3.479 (1H, m, H-1'A),3.689 (m, 1H, H-5), 3.886 (m, 1H, H-1'B), 4.135 (dd, 1H, H-6A,J_(H-6A),H-6B =12.2 Hz, J_(H-6A),H-5 =1.7 Hz), 4.266 (dd, 1H, H-6B,J_(H-6B),H-6A =12.3 Hz, J_(H-6B),H-5 =4.7 Hz), 4.493 (d, 1H, H-1',J_(H-1'),H-2' =7.9 Hz), 4.968 (m, 3H, 2×H-6'+H-2), 5.086 (dd, 1H, H-4,J=H-4, H-5=J_(H-4),H-3 =9.6 Hz), 5.207 (dd, 1H, H- 3, J_(H3-4)=J_(H-3),H-2 =9.4 Hz), 5.782 (m, 1H, H-5').

IR (nujol) 1755 (C═O), 1640 (C═O), 1220, 1040 (C--O--C) cm⁻¹.

¹³ C NMR (75 MHz, CDCl₃) δ 20.559 (OCOCH₃) 25.128 (C-3'), 28.845 (C-2'),33.268 (C-4'), 62.127 (C-6), 68.699, 69.834, 69.852, 71.496, 71.868,72.987 (C-1', C-2, C-3, C-4, C-5), 100.826 (C-1), 114.582 (C-6'),138.437 (C-5'), 169.100, 169.259, 170.132, 170.160 (OCOCH₃).

5'-Hexenyl β-D-Glucopyranoside 5

In a 250-mL round-bottom flask equipped with a magnetic stirring bar,nitrogen inlet and bent-tube adapter were placed 4 (4.29 g, 9.97 mmol),methanol (anhydrous 100 mL) and sodium methoxide (0.5N in methanol, 1.0mL) and stirred at room temperature for 1.25 h. Then it was treated with2.0 g of Bio-Rad ion exchange resin AG-50W-X8 and stirred for 10 min.The reaction mixture was filtered, washed with methanol, and the solventremoved. The residue was dried under vacuum 0.1 mm Hg/18 h to provide 5(2.56 g, 98% yield).

¹ H NMR (300 MHz, D₂ O) δ 1.483 (m, 2H, H-3'), 1.666 (m, 2H, H-2'),2.115 (dd, 2H, H-4', J_(H-4'),H-5' =13.7 Hz, J_(H-4'),H-3' =6.9 Hz),3.270 (t, 1H), 3.469 (m, 3H), 3.718 (m, 2H), 3.930 (m, 2H), (H-1'A,H-1'B, H-2, H-3, H-4, H-5, H-6A, H-6B), 4.464 (d, 1H, H-1, J_(H1),H2=8.0 Hz), 5.038 (m, 2H, H-6'), 5.943 (m, 1H, H-5').

¹³ C NMR (75 MHz, D₂ O) δ 26.713 (C-3'), 30.547 (C-2'), 34.93 (C-4'),63.162 (C-6), 63.162, 72.035, 72.642, 75.468, 78.177 (C-1', C-2, C-3,C-4, C-5), 104.469 (C-1), 116.656 (C-6'), 142.055 (C-5').

IR (nujol) 3470, 3350 (OH), 1640 (C═O), 1080, 1035 (C--O--C).

FAB MS calcd for C₁₂ H₂₂ O₆ (M+H) 263.15. Found 263.28.

5'-Hexenyl,2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 6

In a 250-mL round-bottom flask equipped with a magnetic stirring bar andbent-tube adapter were placed 5 (2.4 g, 9.15 mmol),dimethylaminopyridine (5.58 g, 45 mmol), perfluorooctylpropionic acid(22.49 g, 45.7 mmol), molecular sieves 4 Å powder (500 mg), anhydrousdimethylformamide (25 mL) and Freon®-113(1,1,2-trichlorotrifluoroethane, 25 mL). The mixture was cooled to ˜0°C. and dicyclohexylcarbodiimide (11.33 g, 54.9 mmol) suspended in DMFand Freon®-113 (25 mL+25 mL) was added to the above mixture. Thecontents were stirred at room temperature for 25 h. The mixture wasfiltered and the residue chromatographed on silica gel (325 g, 1:12ethyl acetate/Freon®-113) to provide 6 (14.34 g, 72.6% yield).

¹ H NMR (300 MHz, CDCl₃ +Freon®-113) 1.2-2.0 (bm, 4H, H-2', H-3'), 2.061(m, 2H, H-4'), 2.599 (m, 16H, 4 X--COCH₂ CH₂ (CF₂)--), 3.494 (m, 1H,H-1'A), 3.725 (br m, 1H, H-5), 3.919 (m, 1H, H-1'B), 4.179 (dd, 1H,H-6A, J_(H-6A),H-6B =12.4 Hz, J_(H-6A),H-5 =1.7 Hz), 4.480 (dd, 1H,H-6B, J_(H-6B),H-6A =12.5 Hz, J_(H-6B),H-5 =4.0 Hz), 4.537 (d, 1H, H-1,J_(H-1),H-2 =7.9 Hz), 4.96 (m, 2H, H-6'), 5.074 (dd, 1H, H-2,J_(H-2),H-1 =8.7 Hz), 5.185 (dd, 1H, H-4, J_(H-4),H-3 =J_(H-4),H-5 =9.6Hz), 5.289 (dd, 1H, H-3, J_(H-3),H-4 =J_(H-3),H-2 =9.3 Hz), 5.779 (m,1H, H-5').

¹³ C NMR (75 MHz, CDCl₃ +Freon®-113) δ 24.485 (C-3'), 29.068 (C-2'),33.057 (C-4'), 62.244 (C-6), 69.010, 70.278, 72.187, 72.409, 73.919(C-1', C-2, C-3, C-4, C-5), 100.59 (C-1), 114.414 (C-6'), 137.836(C-5'), 169.857, 170.306, 170.953, 171.102 (4×OCOCH₂ CH₂).

IR (KBr) 1755 (OCOR), 1200 (--CF₂ --), 1150 cm⁻¹.

FAB MS calcd for C₅₆ H₃₄ F₆₈ O₁₀ (M+H) 2059.8. Found 2059.6

4'-(Carboxy)Butyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 7

In a 1-liter, 3-necked, round-bottom flask equipped with an overheadmechanical stirrer, reflux condenser and nitrogen inlet were placed 6(13.34 g, 6.18 mmol), Aliquat® 336 (1.50 g, Aliquat® 336 is a phasetransfer catalyst available from Aldrich Chemical Co.), glacial aceticacid (45 mL), 1,1,2-trichlorotrifluoroethane (150 mL), hexane (150 mL)and cooled to ˜50° C. in an ice-bath. Meanwhile potassium permanganate(23.706 g, 150 mmol) was dissolved in 300 mL of water and then addedslowly to the above mixture with vigorous stirring. After the additionhas been completed, the contents were stirred in the same bath at roomtemperature for 24 h. The excess potassium permanganate was decomposedby the addition of sodium sulfite (30 g) in small portions (cooling maybe required). After stirring the contents for 15 min, the reactionmixture was acidified with 1:1 hydrochloric acid/water while cooling themixture in waterbath (˜20°C.). The reaction mixture was diluted withethyl ether (200 mL) and then poured into a separatory funnel (use brineto break up emulsions if necessary). The organic layer was washed withwater, brine and dried over magnesium sulfate. The residue was driedunder vacuum (1 mm) for 3 days (to make it free from acetic acid) toprovide 7 (12.37 g, 92% yield) as white solid which was used in thefollowing step without further purification.

¹ H NMR (300 MHz, Acetone-D₆ +Freon®-113) δ 1.0-1.8 (m, 4H, H-2'+H-3'),2.284 (m, 2H, H-4'), 2.599 (m, 16H, 4×OCOCH₂ CH₂ CF₂ --), 3.583 (m, 1H,H-1'A), 3.897 (m, 1H, H-1'B), 4.045 (m, 1H, H-5), 4.225 (dd, 1H, H-6A,J_(H-6A),H-6B =12.4 Hz, J_(H-6A),H-5 =1.8 Hz), 4.470 (dd, 1H, H-6B,J_(H-6B),H-6A =12.4 Hz, J_(H-6B),H-5 =4.2 Hz), 4.817 (d, 1H, H-1, J=8Hz), 5.029 (dd, 1H, H-2, J_(H-2),H-3 =9.5 Hz, J_(H-2),H-1 =8.0 Hz),5.219 (dd, 1H, H-4, J_(H-4),H-5 =J_(H-4),H-3 =9.7 Hz), 5.39 (dd, 1H,H-3, J_(H-3),H-2 =9.4 Hz).

4'(N-Oxysuccinimidylcarbonyl)Butyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 8

In a 100-mL round-bottom flask equipped with magnetic stirring bar,nitrogen inlet, and bent-tube adapter were placed 7 (1.985 g, 0.91mmol), powdered molecular sieves 4 Å (50 mg), dimethylaminopyridine (167mg, 1.36 mmol), N-hydroxysuccinimide (156 mg, 1.36 mmol), acetone (10mL) and Freon®-113 (1,1,2-trichlorotrifluoroethane, 15 mL) and stirredat 0° C. To the above mixture was added dicyclohexylcarbodiimide (309mg, 1.5 mmol) in acetone (5 mL). The contents were then allowed to warmup to room temperature and further stirred for 3 h. The reaction mixturewas then filtered and the residue after removal of solvent waschromatographed (silica gel 70 g, 1:5 ethyl acetate/Freon®-113) toprovide 8 (902 mg, 43.6%). The reaction is almost quantitative by TLC(thin layer chromatography). However, the yield varies from experimentto experiment due to instantability of 8 during chromatography.

¹ H NMR (300 MHz, CDCl₃ +Freon®-113) δ0 0.8-2.0 (m, 4H, H-2+H-3'), 2.593(m, 18H, H-4'+4×--COCH₂ CH₂ CF₂ --), 2.807 (s, 4H, succinimidyl Hz),3.595 (m, 1H, H-1'A), 3.748 (m, 1H, H-5), 3.926 (m, 1H, H-1'B), 4.182(dd, 1H, H-6A, J_(H-6A),H-6B =11.1 Hz, J_(H-6A),5 =1.7 Hz), 4.467 (dd,1H, H-6B, J_(H-6A),H-6B =12.5 Hz, J_(6B),H5 =3.9 Hz), 4.591 (d, 1H, H-1,J_(H-1),H-2 =8.0 Hz), 5.082 (dd, 1H, H-2, J_(H2),H3 =J_(H2),H1 =8.7 Hz),5.188 (dd, 1H, H-4, J_(H4),H3 =J_(H4),H5 =9.6 Hz), 5.290 (dd, 1H, H-3,J_(H3),H4 =J_(H3),H2 =9.3 Hz).

4'-Oxopentyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 9

In a 50-mL round-bottom flask with a magnetic stirrer bar, bent-tubeadapter and gas inlet was placed 6 (0.5 g, 0.23 mmol) in dichloromethane(5 mL) and Freon®-113 (10 mL). The resulting solution was cooled to 0°C. and ozone was passed through the solution for 20 min, the excessozone flushed out by passing nitrogen and the ozonide decomposed by theaddition of thiourea (454 mg, 0.6 mmol) in methanol (10 mL) and stirredfor 1 h at room temperature. The contents were diluted with water (100mL) and extracted with Freon®-113. The organic extract was dried andresidue after removal of solvent chromatographed (silica gel 30 g, 1:10ethyl acetate/Freon®-113), to provide 9 (110 mg, 21% yield).

¹ H NMR (300 MHz; CDCl₃ +Freon®-113) δ 1.622 (m, 4H, H-2'+H-3'), 2.606(m, 18H, --COCH₂ CH₂ CF₂ --+H-4'), 3.519 (m, 1H, H-1'A), 3.731 (m, 1H,H-5), 3.905 (m, 1H, H'-1B) 4.186 (br d, 1H, H-6A, J_(H-6A),H-6B =11.8Hz), 4.461 (dd, 1H, H-6B, J_(H-6B),H-6A =12.5 Hz, J_(H-6B),H-5 =4.0 Hz),4.542 (d, 1H, H-1, J_(H-1),H-2 =8.0 Hz), 5.07 (dd, 1H, H-2, J_(H-1),H-2=J_(H-2),H-3 =8.6 Hz), 5.178 (dd, 1H, H-4, J_(H-4),H-3 =J_(H-4),H-5 =9.5Hz), 5.285 (dd, 1H, H-3, J_(H-3),H-4 =J_(H-3),H-2 =9.3 Hz), 9.58 (s, 1H,CHO).

5',6'-Epoxyhexyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 10

In a 100-mL round-bottom flask equipped with magnetic stirring bar,nitrogen inlet and bent-tube adapter was placed 6 (3.1 g, 1.44 mmol) ina mixture of dichloromethane (18 mL) and Freon®-113 (18 mL) andm-chloroperbenzoic acid (m-CPBA) (85%, 0.65 g, 3.19 mmol). The contentswere stirred at room temperature for 4 h. The reaction was incomplete.An additional amount (0.65 g) of m-CPBA was added, and the contentsstirred at room temperature for 18 h. The reaction mixture was cooled to0° C., and the excess m-CPBA was decomposed by the addition of sodiumsulfite (5.17 g, 41 mmol) in water (15 mL). The contents were furtherstirred for 30 min, extracted with a mixture of Freon®-113 anddichloromethane, and the residue chromatographed (silica gel 90 g, 1:10ethyl acetate/Freon®-113) to provide 10 (2.4 g, 77% yield).

¹ H NMR (300 MHz; CDCl₃ +Freon®-113) δ 1.496-1.622 (m, 6H, H-2', H-3',H-4'), 2.606 (m, 18H, --OCOCH₂ CH₂ CF₂ --, H-6'), 2.859 (m, 1H, H-5'),3.519 (m, 1H, H-1'A), 3.731 (m, 1H, H-5), 3.905 (m, 1H, H-1'B), 4.186(br d, 1H, H-6A, J_(H-6A),H-6B =11.8 Hz), 4.461 (dd, 1H, H-6B,J_(H-6B),H-6A =12.5 Hz, J _(H-B6),H-5 =4.0 Hz), 4.542 (d, 1H, H-1,J_(H-1),H-2 =8.0 Hz), 5.070 (dd, 1H, H-2, J_(H-1),H-2 =J_(H-2),H-3 =8.6Hz), 5.178 (dd, 1H, H-4, J_(H-4),H-3 =J_(H-4),H-5 =9.5 Hz), 5.285 (dd,1H, H-3, J_(H-3),H-4 =J_(H-2),H-3 =9.3 Hz).

¹³ C NMR (75 MHz; CDCl₃ +Freon®-113) δ 22.798, 22.822 (C-3') 29.342,29.392 (C-2'), 32.277, 32.291 (C-4'), 46.734 (C-6'), 52.031, 52.065(C-5'), 62.232 (C-6), 68.985, 70.006, 70.251, 72.181, 72.350, 73.902,77.201 (C-2, C-3, C-4, C-5, C-1'), 101.043, 101.090 (C-1), 169.936,170.298, 170.836, 171.104. (The compound is a mixture ofdiastereoisomers at C-5'.)

IR (nujol) 1755 (OCOR), 1250-1150 (CF₂).

5'-Hydrazinocarbonylpentyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 11

In a 200-mL round-bottom flask equipped with reflux condenser, magneticstirring bar and inlet were placed under oxygen and moisture-freeconditions compound 8 (1.5 g, 0.66 mmol), Freon®-113 (37.5 mL),trifluoroethanol (37.5 mL; trifluoroethanol is a reproductive toxin),and cooled to 0° C. Anhydrous hydrazine (0.105 mL), was added slowly andthe contents allowed to warm up to room temperature and stirred for 3 h.(TLC 1:5 ethyl acetate/Freon®-113). The contents were made free ofsolvents and the residue chromatographed (silica gel 80 g, 10%trifluoroethanol in Freon®-113) to provide 11 (1.00 g, 69.8% yield).

¹ H NMR (300 MHz; CDCl₃ +Freon®-113) δ 0.85-2.05 (m, 4.0H, H-2', H-3'),2.160 (m, 1H, H-4'), 2.573 (m, 16H, --OCOCH₂ CH₂ CF₂ --), 3.537 (m, 1H,H-1'A), 3.733 (m, 1H, H-5), 3.909 (m, 1H, H-1'B), 4.123 (br d, 1H, H-6A,J_(H-6A),H-6B =10.0 Hz), 4.47 (dd, 1H, H-6B, J_(H-6B),H-6A =12.6 Hz,J_(H-6B),H-5 =4.0 Hz), 4.53 (d, 1H, H-1, J_(H-1),H-2 =8.0 Hz), 5.093(dd, 1H, H-2, J_(H-2),H-3 =J_(H-2),H-1 =8.6 Hz), 5.187 (dd, 1H, H-4,J_(H-4),H-5 =9.6 Hz), 5.299 (dd, 1H, H-3, J_(H-3),H-4 =J_(H-3),H-2 =9.3Hz).

¹³ C NMR (75 MHz; CDCl₃ +Freon®-113) δ 22.010, (C-3'), 28.322 (C-2'),33.312 (C-4'), 61.652 (C-6), 69.620, 71.126, 71.839, 73.314, (C-1', C-2,C-3, C-4, C-5), 100.640 (C-1), 169.765, 169.810, 170.281, 170.635,173.149 (433 --OCOCH₂ CH₂, CONHNH₂).

IR (KBr) 3340 (NH--NH₂), 1750 (--OCOCH₂ --), 1655, 1250-1200 (CF₂).

FAB MS calcd for C₅₅ H₃₄ F₆₈ N₂ O₁₁ (M+H) 2191.11. Found 2190.20.

5'-(N-Benzyloxycarbonyl)Aminopentanol 13

In a 500-mL round-bottom flask equipped with magnetic stirring bar,bent-tube adapter and nitrogen inlet were placedN-benzyloxycarbonylsuccinimide (23.3 g, 93.5 mmol), absolute ethylalcohol (250 mL) and 5-aminopentanol 12 (9.65 g, 93.5 mmol). Thecontents were stirred at room temperature for 23 h. The solvent wasremoved and the residue chromatographed (silica gel 325 g, 20:1:20 ethylacetate/ethyl alcohol/hexane) to provide 13 (14.053 g, 63.3%).

¹ H NMR (300 MHz; CDCl₃) δ 1.401 (m, 2H, H-3), 1.557 (m, 4H, H-2, H-4),1.743 (s, 1H, OH), 3.023 (q, 2H, H-5, J=6.6 Hz), 3.632 (q, 2H, H-1,J=5.8 Hz), 4.826 (br s, 1H, NH), 5.089 (s, 2H, --OCH₂ Ph), 7.350 (s, 5H,aromatic).

¹³ C NMR (75 MHz; CDCl₃) δ 22.889 (C-3), 29.750 (C-4), 32.215 (C-2),40.96 (C-5), 62.553 (C-1), 66.593 (OCH₂ Ph), 127.987, 128.425, 136.621(aromatic carbons), 156.442 (OCOO).

IR (KBr) 3400 (--NH--), 3340 (--OH), 1690 (--NHCOO), 1535, 1260, 1020.

FAB MS calcd for C₁₃ H₁₉ NO₃ (M+H) 238.14 Found 238.08

5'-(N-Benzyloxycarbonyl)Aminopentyl2,3,4,6-Tetra-O-Acetyl-β-D-Glucopyranoside 14

In a 1-liter round-bottom flask equipped with an overhead mechanicalstirrer, reflux condenser and addition funnel were placed 13 (14.053 g,59 mmol), silver carbonate (16.54 g, 60 mmol), calcium sulfate (10 g)and dichloromethane (200 mL). To the vigorously stirred solution wasadded 3 (12.3 g, 30 mmol) in 100 mL of dichloromethane over a 2 hperiod. The mixture was further stirred for 40 h at room temperature,filtered through a Celite pad and the residue after removal of solventwas dissolved in a mixture (1:1) of toluene and nitromethane (200 mL).To the above solution was added 500 mg of mercuric bromide and thecontents were heated at 50° C. (bath) over a 24 h period. The solventwas removed, residue dissolved in dichloromethane and the organicextract washed with sodium thiosulfate. The organic extract after dryingand removal of solvent was chromatographed (silica gel 500 g, 2:3 ethylacetate/hexane) to provide 14 (5.849 g, 34.4%).

¹ H NMR (300 MHz; CDCl₃) δ 1.355 (m, 2H, H-3') 1.507 (m, 1H, H-4'),1.586 (m, 1H, H-2'), 2.003, 2.024, 2.024, 2.078 (4 s, 12H, 4×OCOCH₃),3.176 (br q, 2H, H-5', J=6.6 Hz), 3.474 (m, 1H, H-1'A), 3.68 (m, 1H,H-5), 3.86 (m, 1H, H-1B), 4.138 (dd, 1H, H-6A, J_(H-6A),H-6B =12.3 Hz,J_(H-6A),H-5 =2.4 Hz), 4.258 (dd, 1H, H-6B, J_(H-6B),H-6A =12.3 Hz,J_(H-6B),H-5 =4.7 Hz), 4.479 (d, 1H, H-1, J_(H-1),H-2 =7.9 Hz), 4,841(m, 1H, --NH--), 4.975 (dd, 1H, H-2, J_(H-2),H-3 =9.4 Hz, J_(H-1),H-2=8.0 Hz), 5.079 (dd, 1H, H-4, J_(H-3),H-4 =J_(H-4),H-5 =9.6 Hz), 5.088(s, 1H, --OCH₂ Ph), 5.200 (dd, 1H, H-3, J_(H-2),H-3 =J_(H-3),H-4 =9.4Hz), 7.347 (s, 5H, aromatic).

¹³ C NMR (75 MHz, CDCl₃) δ 20.545, 20.655 (OCOCH₃), 23.072 (C-3'),29.986 (C-4'), 29.564 (C-2'), 40.976 (C-5'), 62.043 (C-6), 66.575(--OCH₂ Ph), 68.642, 69.692, 71.456, 71.864, 72.92 (C-1', C-2, C-3, C-4,C-5), 100.762 (C-1), 127.993, 128.436, 136.621 (aromatic), 156.349(--NHCOO--), 169.161, 169.266, 170.121, 170.490 (4×--OCOCH₃).

IR (KBr) 3350 (--NH--), 1750 (--OCOCH₃), 1730, 1685 (--NHCOO--), 1540,1250-1220, 1050, 1030.

FAB MS calcd for C₂₇ H₃₇ NO₁₂ (M+H) 568.24. Found 568.10.

5'-(N-Benzyloxycarbonyl)Aminopentyl β-D-Glucopyranoside 15

In a 300-mL round-bottom flask equipped with magnetic stirrer bar,septum inlet and bent-tube adapter were placed 14 (3.913 g, 6.89 mmol),methanol (dry; 150 mL) and 0.5N sodium methoxide (2.5 mL) in methanol.The pH of the solution should be >10. The contents were stirred for 3 h(TLC 14:4:1 ethyl acetate/ethyl alcohol/water indicated completion ofthe reaction). The mixture was neutralized with 1 g of Bio-Rad H⁺ ionexchange resin AG-50W-X8 and stirred for 10 min. The mixture wasfiltered and washed with methanol. The filtrate was taken to dryness toprovide 15 (2.521 g, 91.5% yield).

¹ H NMR (300 MHz; D₂ O) δ 1.092 (m, 2H, H-3'), 1.232 (m, 2H, H-4'),1.346 (m, 2H, H-2'), 2,850 (br t, 2H), 2.972 (t, 1H), 3.125 (m, 3H),3.406 (m, 2H), 3.611 (m, 2H), (H-2, H-3, H-4, H-5, H-6, H-1', H-5'),4.154 (d, 1H, H-1, J_(H-1),H-2 =7.9 Hz), 4.83 (s, 2H, --OCH₂ Ph), 7.146(s, 5H, aromatic).

¹³ C NMR (75 MHz; D₂ O) δ 22.296 (C-3'), 28.408 (C-4'), 28.610 (C-2'),40.456 (C-5'), 60.907 (C-6), 66.646 (--OCH₂ Ph), 69.754, 69.754, 70.213,73.187, 75.871 (C-2, C-3, C-4, C-5, C-1'), 102.224 (C-1), 127.554,128.159, 128.625, 136.542 (aromatic), 158.095 (--NHCOO--).

IR (KBr) 3320 (--OH,NH--), 1685 (--NHCOO--), 1540, 1260 (C--O), 1030(C--O).

FAB MS calcd for C₁₉ H₂₉ NO₈ (M+H) 400.20 Found 400.03.

5'-(N-Benzyloxycarbonyl)Aminopentyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 16

In a 100-mL round-bottom flask equipped with a magnetic stirrer bar,septum inlet and bent-tube adapter were placed 15 (600 mg, 1.5 mmol),molecular sieves 4 Å (150 mg), dimethylaminopyridine (911 mg, 7.5 mmol),perfluorooctylpropionic acid (4.69 g, 7.5 mmol), anhydrous dimethylformamide (2.5 mL) and anhydrous Freon®-113 (7.5 mL). The above mixturewas stirred at 0° C. and dicyclohexylcarbodiimide (1.857 g, 9 mmol) in 5mL of anhydrous dimethylformamide was added to the above mixture. Thecontents were then stirred at room temperature for 20 h. The reactionmixture was filtered, washed with 1:1 Freon®-113/dichloromethane mixture(3×50 mL) and the filtrate concentrated. The residue after solventremoval was chromatographed (silica gel 200 g, 1:5 ethylacetate/Freon®-113 to provide 16 (2.984 g, 86.6% yield).

¹ H NMR (300 MHz; CDCl₃ +Freon®-113) δ 1.401 (m, 2H, H-3'), 1.527 (m,2H, H-4'), 1.617 (m, 2H, H-2'), 2.541 (m, 16H, --OCOCH₂ CH₂ (CF₂)₇ CF₃),3.198 (br q, 2H, H-5', J=6.6 Hz), 3.509 (m, 1H, H-1'A), 3.719 (br d, 1H,H-5), 3.88 (m, 1H, H-1'B), 4.185 (br d, 1H, H-6A, J_(H-6A),H-6B =11.1Hz), 4.471 (dd, 1H, H-6B, J_(H-6B),H-6A =12.4 Hz, J_(H-6B),H-5 =3.8 Hz),4.530 (d, 1H, H-1, J_(H-1),H-2 =8.0 Hz), 4.733 (m, 1H, --NH--), 5.066(dd, 1H, H-2, J_(H-2),H-3 =J_(H-1),H-2 =9.0 Hz), 5.096 (s, 2H, --OCH₂Ph), 5.179 (dd, 1H, H-4, J_(H-4),H-3 =J_(H-4),H-5 =9.6 Hz), 5.284 (dd,1H, H-3, J_(H-3),H-4 =J_(H-3),H-2 =9.4 Hz), 7.337 (s, 5H, aromatic).

¹³ C NMR (300 MHz; CDCl₃ +Freon®-113) δ 22.932, (C-3'), 28.895 (C-4'),29.590 (C-2'), 40.811 (C-5'), 61.994 (C-6), 66.622 (--OCH₂ Ph), 68.558,69.956, 71.611, 71.890, 73.383 (C-2, C-3, C-4, C-5, C-1'), 100.547(C-1), 127.554, 128.498, 136.556 (aromatic), 158.095 (--NHCOO--),169.936, 170.298, 170.836, 171.104 (4×--OCOCH₂ CH₂ CF₂ --).

IR (KBr) 3380 (--NH--), 1755 (--OCOCH₂), 1695 (--NHCOO--), 1540,1250-1200, (--C--O--CF--), 1150 (C--O).

FAB MS calcd for C₆₃ H₄₁ F₆₈ NO₁₂ (M+H) 2296.16. Found 2296.00.

5'-Aminopentyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 17

In a 50-mL round-bottom flask equipped with magnetic stirrer bar, septuminlet and bent-tube adapter were placed 16 (229 mg, 0.1 mmol), palladiumblack (50 mg), trifluoroethanol (2 mL) and Freon®-113 (18 mL) andstirred at room temperature. The mixture was then exposed to hydrogenusing hydrogen balloon for 3 h. At this point, the reaction was complete(TLC 10% methanol in Freon®-113). The reaction mixture was filteredthrough a Celite pad and the filtrate concentrated to provide 17 (204mg, 94% yield).

¹ H NMR (300 MHz; CF₃ CD₂ OD+Freon®-113) δ 1.376 (m, 2H, H-3'), 1.595(m, 2H, H-4'), 1.660 (m, 2H, H-2'), 2.566 (m, 16H, OCOCH₂ CH₂ CF₂ --),2.937 (br s, 2H, H-5'), 3.540 (m, 1H, H-1'A), 3.815 (m, 1H, H-1'B),4.207 (d, 1H, H-6A, J_(H-6A),H-6B =12.1 Hz), 4.318 (d, 1H, H-6B,J_(H-6B),H-6A =12.0 Hz), 4.513 (d, 1H, J_(H-1),H-2 =6.9 Hz), 4.513 (d,1H, H-1, J_(H-1),H-2 =8.2 Hz), 4.967 (dd, 1H, H-2, J_(H-2),H-3=J_(H-1),H-2 =8.2 Hz), 5.139 (dd, 1H, H-4, J_(H-4),H-3 =J_(H-4),H-5 =8.6Hz), 5.273 (dd, 1H, H-3, J_(H-3),H-4 =J_(H-3),H-2 =9.3 Hz).

IR (KBr) 3420 (--NH₂), 1750 (--OCOCH₂ --), 1250-1200 (--CF--), 1150(C--O).

FAB MS calcd for C₅₅ H₃₅ F₆₈ NO₁₀ (M+H) 2162.12. Found 2162.32.

4-Pentenylamine (19)

A mixture of lithium aluminum hydride (10.3 g, 13.6 mmol) and diethylether (500 mL) were cooled to ˜5° C. (ice-bath). To the above stirredsuspension was added slowly a solution of 4-pentenenitrile (11.0 g, 136mmol) in 50 mL anhydrous ether. The reaction mixture was allowed to warmup to room temperature and further stirred for 2 h. The excess reagentwas quenched with sodium sulfate (solid), filtered, dried over sodiumsulfate and filtered again. The filtrate was distilled through aVigreaux column to give 7.16 g of 19 as colorless liquid, bp 107°-111°C.

¹ H NMR (300 MHz; CDCl₃) δ 1.433 (s, 2H, NH₂), 1.548 (quint, 2H, H-2,H=7.3 Hz), 2.099 (quart, 2H, H-3, J=6.9 Hz), 2.709 (t, 2H, H-1, J=7.1Hz), 4.996 (m, 2H, H-5), 5.819 (m, 1H, H-4).

IR (NaCl) 3280 (NH₂), 1640 (CH═CH₂) cm⁻¹.

N-4'-Pentenyl Gluconamide 20

Gluconolactone (18) (1.34 g, 7.5 mmol), 4-pentenylamine (0.85 g, 10mmol), acetronitrile (34 mL) and water (11 mL) were combined and heatedat 60° for 2 h. The mixture was made free of solvent, residue dissolvedin water and lyophilized to give 1.94 g of 20. The crude product wastreated with pyridine (29.8 mL, 369 mmol) and acetic anhydride (17.4 mL,185 mmol) and stirred at room temperature for 18 h. It was poured intomixture of water and ice and then extracted with dichloromethane (3×50mL). The combined dichloromethane layer was washed with 1N HCl (150 mL,ice-cold), 50 mL of saturated sodium bicarbonate, 50 mL of brine andthen dried over magnesium sulfate. The residue after filtration andremoval of solvent furnished 2.35 g, 71% yield, or pure 21 after flashchromatography (silica gel 130 g, 1:2 ethyl acetate/hexane).

¹ H NMR (CDCl₃) δ 1.602 (quint, 2H, H-2', J=7.3 Hz), 2.057 (s, 3H,OCOCH₃), 2.098 (s, 3H, --OCOCH₃), 2.117 (s, 3H, --OCOCH₃), 2.209 (s, 3H,--OCOCH₃), 2.2 (m, 2H, H-3'), 3.276 (br q, 2H, H-1', J=6.2 Hz), 4.131(dd, 1H, H-6A, J_(H-6A),H-5 =5.4 Hz, J_(H-6A),H-6B =12.2 Hz), 4.319 (dd,1H, H-6B, J_(H-6B),H-5 =4.0 Hz, J_(H-6B),H-6A =12.3 Hz), 5.010 (m, 2H,H-5'), 5.065 (m, 1H, H-5), 5.292 (d, 1H, H-2, J_(H-2),H-3 =5.5 Hz),5.447 (dd, 1H, H-4, J_(H-4),H-3 =5.0 Hz, J_(H-4),H-5 =6.4 Hz), 5.684(dd, 1H, H-3, J_(H-3),H-2 =5.2 Hz, J_(H-3),H-4 =5.2 Hz), 5.788 (m, 1H,H-4'), 6.134 (br t, 1H, NH).

¹³ C NMR (75 MHz, CDCl₃), 20.245, 20.477 (OCOCH₃), 28.400 (C-2'), 30.840(C-3'), 38.923 (C-1'), 61.462 (C-6), 68.779 (C-5), 69.022 (C-3), 69.388(C-4), 71.600 (C-2), 115.218 (C-5'), 137.359 (C-4').

IR (KBr) 3360, 3250 (--NH), 1750 (--COCH₃), 1660, 1670 (--CONH--), 1530(--CONH--), 1220 (ester) cm⁻¹.

Pentaacetate 21 was saponified as follows: the pentacetate 21 (2.11 g,4.5 mmol) was dissolved in 45 mL of dry methanol and treated with sodiummethoxide (0.45 mL, 0.5M) an stirred at room temperature for 1 h. To themixture was then added acidic resin AG-50W-X8 (0.94 g) and stirred for10 min. The mixture was filtered and evaporated to provide 20 (1.21 g,72% overall yield) as white solid.

¹ H NMR (D₂ O) δ 1.615 (quint, 2H, H-2', J=7.3 Hz), 2.077 (q, 2H, H-3',J=7.2 Hz), 3.243 (t, 2H, H-1', J=6.9 Hz), 3.715-4.044 (m, 4H, H-3, H-4,H-5, H-6A, H-6B), 4.272 (d, 1H, H-2, J_(H-2),H-3 =3.6 Hz), 5.035 (m, 2H,H-5'), 5.874 (m, 1H, H-4').

¹³ C NMR (D₂ O) (¹ H decoupled) δ 27.671 (C-2'), 30.295 (H-3'), 38.694(H-1'), 62.842, 70.549, 71.455, 72.247, 73.441 (C-2, C-3, C-4, C-5,C-6), 114.857 (C-5'), 138.924 (C-4'), 173.848 (C-1).

IR (KBr) 3320 (OH),1650 (--CONH--), 1540 (--CONH--) cm⁻¹.

N-4'-Pentenyl 2,3,4,5,6-Penta-O-(3-Perfluorooctyl)Propionyl Gluconamide22

In a 100-mL round-bottom flask were placed compound 20 (0.26 g, 1 mmol),powdered molecular sieves (0.4 g), N,N-dimethylaminopyridine (0.76 g,6.3 mmol), perfluorooctylpropionic acid (3.1 g, 6.3 mmol), anhydrousdimethylformamide (5 mL), and Freon®-113 (15 mL) and cooled to ˜0° C. Tothe above mixture was added dicyclohexylcarbodiimide (1.6 g, 7.5 mmol)in 10 mL of Freon®-113 and stirred for 18 h at room temperature. Themixture was filtered and the residue after removal of solvent waschromatographed (silica gel, 1:10, ethyl acetate/Freon®-113) to provide22 as white solid (3.11 g).

¹ H NMR (300 MHz; CDCl₃ +Freon®-113 1:1) δ 1.05-2.15 (m, 27H, expected4H, H-2', H-3' and impurity), 2.613 (m, 27H, expected 20H, --COCH₂ CH₂--C₈ F₁₇), 3.725 (m, 1.2H impurity), 3.967 (m, 1.3H, impurity), 4.22(dd, 1H, H-6A, J_(H-6A),H-6B =12.4 Hz, J_(H-6A),H-5 =6.0 Hz), 4.466 (dd,1H, H-6B, J_(H-6B),H-6A =12.3 Hz, J_(H-6B),H-5 =3.3 Hz), 5.01 (m, 2H,H-5'), 5.115 (m, 1H, H-5), 5.406 (d, 1H, H-2, J_(H-2),H-3 =5.3 Hz),5.532 (t, 1H, J=5.7 Hz), 5.784 (m, 2H, H-3 and H-4'), 6.105 (br t, 1H,NH).

IR (KBr) 3440 (NH), 1750 (--COO--), 1650 (--CONH--), 1540 (--CONH--),1200 (CF) cm⁻¹.

FABS MS m/e (M+H) calcd for C₆₆ H₃₆ NO₁₁ F₈₅ 2634.9. Found 2634.8.

N-(4',5'-Epoxy)Pentyl 2,3,4,5,6-Petna-O-(3-Perfluorooctyl-PropionylGluconamide 23

In a 250-mL round-bottom flask equipped with magnetic stirring bar andbent-tube adapter were placed compound 22 ((3.0 g, 1.13 mmol),dichloromethane (50 mL), Freon®-113 (50 mL), and m-chloroperbenzoic acid(1.15 g, 85%, 5.65 mmol) and stirred at room temperature for 18 h. Itwas then cooled to ˜5° C. and slowly treated with sodium sulfite (9.63 gin 52 mL of water) and further stirred for 0.5 h. It was then taken upin a separatory funnel and washed successively with cold saturatedsodium bicarbonate, cold brine and the organic extract after dryingremoval of solvent was chromatographed (silica gel 1:7 ethylacetate/Freon®-113) to provide 23 (2.5 g, 85% yield) as white solid.

¹ H NMR (300 MHz; CDCl₃, Freon®-113 1:1) δ 0.8-2.2 (m, 4H, H-2', H-3'),2.719 (m, 23H, --OCOCH₂ CH₂ (CF₂)CF₃, H-4', H-5'), 3.16, 3.322, 3.473,3.62 (4 m, 2H, H-1'), 4.25 (dd, 1H, H-6A, J_(H-6A),H-6B =12.3 Hz,J_(H-6A),H-5 =6.3 Hz), 4.507 (br d, 1H, H-6B, J=8.8 Hz), 5.143 (br q,1H, H-5), 5.469 (d, 0.5H, H-2A, J_(H-2A),H-3 =4.8 Hz), 5.495 (d, 0.5H,H-2B, J_(H-2B),H-3 =4.8 Hz), 5.588 (t, 1H, H-4, J=5.7 Hz), 5.830 (t, 1H,H-3, J=5.3 Hz), 6.790-6.969 (2 br t, 1H, NH).

IR (KBr) 1750 (ester), 1670 (amide I), 1540 (amide II), 1200 (CF) cm⁻¹.

FAB MS m/e (M+H) calcd for C₆₆ H₃₆ NO₁₂ F₈₅ 2650.9. Found 2651.6.

N-3'-Carboxypropyl 2,3,4,5,6-Penta-O-(3-Perfluorooctyl)PropionylGluconamide 24

In a 100-mL round-bottom flask compound 22 (1.32 g, 0.5 mmol), sodiummetaperiodate (0.21 g, 1.0 mmol), periodic acid (0.23 g, 1 mmol),ruthenium tetroxide (5 mg), Freon®-113 (7 mL), acetronitrile (7 mL), andwater (9 mL) were combined and stirred for 2 h a room temperature. Thereaction mixture was filtered and washed with Freon®-113. The combinedorganic layer was washed with 20 mL of brine, dried over magnesiumsulfate and the filtrate after removal of solvent furnished 24 (1.31 g,99% crude).

¹ H NMR (300 MHz; Acetone-d₆, Freon®-113) δ 1.0-2.1 (m, 26H, expected2H, H-2 impurity), 2.303 (t, 2H, H-3', J=7.2 Hz), 2.69 (m, 27 H,expected 20 H, --OCOCH₂ CH₂ (CF₂)₇ CF₃, 3.236 (m, 2H, H-1'), 3.638 (m,1.2H, imp), 4.013 (m, 1.2H, impurity), 4.313 (dd, 1H, H-6A, J=12.2 Hz,J_(H-6A),H-5 =6.3 Hz), 4.506 (dd, 1H, H-6b, J_(H-6B),H-6A =12.2 Hz,J_(H-6B),H-5 =4.0 Hz), 5.226 (br q, 1H, H-5), 5.416 (d, 1H, H-2,J_(H-2),H-3 =4.6 Hz), 5.612 (t, 1H, H-4, J=5.6 Hz), 5.824 (m, 1H, H-3),7.438 (br d, 1H, impurity), 7.595 (br t, 1H, NH).

IR (KBr) 3340 (MH), 1750 (--COO--), 1680, 1660 (amide I), 1530 (amideII), 1200 (CF) cm⁻¹.

N-3'-(N-Oxysuccinimidylcarbonyl)Propyl2,3,4,5,6-Penta-O-(3-Perfluorooctyl)Propionyl Gluconamide 25

In a 100-mL round-bottom flask were placed compound 24 (1.0 g, 0.38mmol), powdered dry 4 Å molecular sieves (50 mg), dimethylaminopyridine(122 mg, 1.0 mmol), N-hydroxysuccinimide (116 mg, 1.0 mmol), acetone(7.5 mL) and Freon®-113 (7.5 mL) and stirred at ˜5° C. To the abovemixture was added dicyclohexylcarbodiimide (240 mg, 1.16 mmol) and theresulting mixture was stirred at room temperature for 18 h.Dimethylaminopyridine, N-hydroxy succinimidyl ester anddicyclohexylcarbodiimide were again added in the same amount as beforeand stirred for 3 h at room temperature. The reaction mixture was thenfiltered, concentrated and purified by flash chromatography (silica gel,1:3 ethyl acetate/Freon®-113) to give 25 (0.29, 28% yield) as whitesolid.

¹ H NMR (300 MHz; CDCl₃, Freon®-113 1:1) δ 2.050 (t, 2H, H-2'), 2.651(m, 26H, H-3', --COCH₂ CH₂ CF₂ -- and --COCH₂ CH₂ CH₂ COON), 3.345-3.485(m, 2H, H-1'), 4.195 (dd, 1H, H-6A, J_(H-6A),H-6B =12.4 Hz, J_(H-6A),H-5=6.2 Hz), 4.438 (dd, 1H, H-6B, J_(H-6B),H-6A =12.4 Hz, J_(H-6B),H-5 =3.1Hz), 5.167 (m, 1H, H-5), 5.343 (d, 1H, H-2, J_(H-2),H-3 =6.0 Hz), 5.518(dd, 1H, H-4, J_(H-4),H-3 =4.6 Hz, J_(H-4),H-5 =6.6 Hz), 5.728 (dd, 1H,H-3, J=H-3, H-2=5.8 Hz, J_(H-3),H-4 =4.7 Hz), 6.85 (br t, 1H, --NH--).

N-4'-Pentenyl Glucoheptanamide 27

In a 250-mL round-bottom flask equipped with magnetic stirrer andbent-tube adapter were placed glucoheptanolactone 26 (3.7 g, 17.7 mmol),4-pentenylamine 19 (2.0 g, 29.5 mmol), anhydrous dimethylformamide (150mL) and the mixture was stirred at room temperature for 22 h. Thesolvent was removed under high vacuum (0.1 mm, 46°-53° C., bathtemperature) to provide 10 (5.3 g, ˜100% yield) in essentially ˜100%purity and it was used without further purification.

¹ H NMR (D₂ O) δ 1.599 (quint, 2H, H-2', J=7.3 Hz), 2.066 (q, 2H, H-3',J=7.2 Hz), 3.22 (t, 2H, H-1', J=7.0 Hz), 3.59-3.79 (m, 6H, H-3, H-4,H-5, H-6, H-7A, H-7B), 4.204 (d, 1H, H-2, J_(H-2),H-3 =5.4 Hz), 4.99 (m,2H, H-5'), 5.871 (m, 1H, H-4').

IR (KBr) 3330 (--OH), 1650 (amide I), 1540 (amide II) cm⁻¹.

The structure of 27 was further confirmed by its conversion intohexaacetate 28.

¹ H NMR (CDCl₃) δ 1.651 (quint, 2H, H-2', J=7.3 Hz), 2.022-2.216 (5 s+m,20H, 6 OCOCH₃, H-3'), 3.350 (m, 2H, H-1'), 4.076 (dd, 1H, H-7A,J_(H-7A),H-7B =12.5 Hz, J_(H-7A),H-6 =5.4 Hz), 4.229 (dd, 1H, H-7B,J_(H-7B),H-7A =12.5 Hz, J_(H-7B),H-6 =3.0 Hz), 5.027 (m, 2H, H-5'),5.099 (m, 1H, H-6), 5.235 (d, 1H, H-2, J_(H-2),H-3 =3.1 Hz), 5.429 (dd,1H, H-3, J_(H-3),H-2 =3.1 Hz, J_(H-3),H-4 =7.8 Hz), 5.815 (m, 3H, H-4,H-5, H-4'), 6.257 (br t, 1H, NH).

¹³ C NMR (CDCl₃) 20.504, 20.551, 20.627, 20,640, 20.799 (5×OCOCH₃),28.642 (C-2'), 31.009 (C-3'), 38.883 (C-1'), 61.924 (C-7), 68.515 (C-6),68.874, 69.507 (C-4, C-5), 76.576 (C-2), 76.761 (C-3), 115.404 (C-5'),137.520 (C-4').

IR (KBr) 1754 (ester), 1680 (amide I), 1540 (amide II), 1220 (CF) cm⁻¹.

N-4'-Pentenyl 2,3,4,5,6,7-Hexa-O-(3-Perfluorooctyl)PropionylGlucoheptanamide 29

In a 100-mL round-bottom flask were placed compound 27 (0.47 g, 1.6mmol), dimethylaminopyridine (1.5 g, 12 mmol), 4 Å molecular sieves(0.35 g), perfluorooctylpropionic acid (5.9 g, 11.9 mmol), anhydrousdimethylformamide (15 mL), and Freon®-113 (10 mL) and cooled to ˜5° C.To this stirred mixture was added a solution of dicyclohexylcarbodiimide(3.0 g, 14.4 mmol) in 23 mL of Freon®-113, and the resulting solutionwas stirred at room temperature for 18 h. The reaction mixture was thenfiltered, concentrated and chromatographed (silica gel 1:15 ethylacetate/Freon®-113 to provide 29 (3.8 g, 77% yield) as white solid.

¹ H NMR (CDCl₃ +Freon®-113 1:1) δ 1.661 (quint, 2H, H-2', J=7.2 Hz),2.13 (q, 2H, H-3', J=7.1 Hz), 2.626 (m, 24H, --COCH₂ CH₂ C₈ F₁₇), 3.337(m, 2H, H-1'), 4.160 (dd, 1H, H-7A, J_(H-7A),H-7B =12.6 Hz, J_(H-7A),H-6=5.4 Hz), 4.319 (dd, 1H, H-7B, J_(H-7B),H-7A =12.5 Hz, J_(H-7B),H-6 =2.4Hz), 5.027 (m, 2H, H-4'), 5.201 (m, 1H, H-6), 5.257 (d, 1H, H-2,J_(H-2),H-3 =2.3 Hz), 5.794 (m, 1H, H-5'), 5.989 (d, 2H, H-4 and H-5,J=8.7 Hz), 6.238 (br t, 1H, NH).

IR (KBr) 1750 (ester, 1680 (amide I), 1540 (amide II), 1200 (CF) cm⁻¹.

FAB MS m/e (M+H) calcd for C₇₈ H₄₁ NO₁₃ F₁₀₂ 3139.0. Found 3138.9.

N-(4',5'-Epoxy)-Pentyl 2,3,4,5,6-Hexa-O-(3-Perfluorooctyl)PropionylGlucoheptanamide 30

In a 50-mL bottom-round flask were placed compound 29 (1.0 g, 0.32mmol), dichloromethane (8.5 mL), Freon®-113 (8.5 mL) and cooled to ˜5°C. and then m-chloroperbenzoic acid (85%, 0.33 g, 1.6 mmol) was added tothe above mixture. The reaction mixture was stirred at room temperaturefor 18 h. The excess m-chloroperbenzoic acid was decomposed by theaddition of sodium sulfite (11.5 g in 63 mL of H₂ O) at ˜5° C. followedby stirring at room temperature for 0.5 h. The reaction mixture wasdiluted with 25 mL of dichloromethane and 25 mL of Freon®-113 and thecombined organic extract was washed successively with saturated sodiumbicarbonate, brine and then dried over anhydrous sodium sulfate. Theextract after filtration followed by removal of solvent andchromatography furnished 30 (silica gel, 1:12 ethyl acetate/Freon®-113,0.56 g, 56%) as white solid.

¹ H NMR (CDCl₃ /Freon®, 1:1) δ 1.254 (m, 1H, H-2'A, 1.7 (m, 1H, H-2'B),1.797 (m, 1H, H-3'A), 2.087 (m, 1H, H-3'B), 2.510 (m, 3.274, 28H,--COCH₂ CH₂ CF₂ --, H-4', H-5'), 3.274 (m, 1H, H-1'A), 3.561 (m, 1H,H-1B), 4.156 (dd, 1H, H-7A, J_(H-7A),H-7B =12.6 Hz, J=H-7A, H-6=5.5 Hz),4.323 (dd, 1H, H-7B, J_(H-7B),H-7A =12.78 Hz, J_(H-7B),H-6 =2.2 Hz),5.198 (m, 1H, H-6), 5.256 (d, 0.5H, H-2A, J_(H-2A),H-2 =2.3 Hz), 5.264(d, 0.5H, H-2B, J_(H-2B),H-3 =2.3 Hz), 5.508 (m, 1H, H-3), 5.966 (d, 1H,H-4 or H-5, J=6.5 Hz), 5.988 (d, 1H, H-4 or H-5, J=6.7 Hz), 7.013 (brquart, 1H, NH).

IR (KBr) 1750 (ester), 1680 (amide I), 1550 (amide II), 1200 (CF) cm⁻¹.

FAB MS m/e (M+H) calcd for C₇₈ H₄₁ NO₁₄ F₁₀₂ 3155.0. Found 3155.4.

N-3'-Carboxypropyl 2,3,4,5,6,7-Hexa-O-(3-Perfluorooctyl)PropionylGlucoheptanamide 31

In a 250-mL round-bottom flask equipped with magnetic stirring bar andbent-tube adapter were placed compound 29 (2.6 g, 0.85 mmol), Aliquat®336 (0.21 g), acetic acid (6.3 mL), hexane (42 mL), Freon® (42 mL) andcooled to ˜5° C. in an ice bath. To the above stirred mixture was addedpotassium permanganate (3.3 g, 20.9 mmol) in 83 mL of water. Thereaction mixture was then allowed to stir at room temperature for 18 h.It was cooled to ˜5° C. and quenched with sodium sulfite (8.3 g, 66mmol). After stirring the contents at room temperature for 10 min, thereaction mixture was acidified with 22 mL of 6N HCl. The organic layerwas diluted with ether and the aqueous layer was extracted three timeswith ether/Freon®-113 mixture (6:1). The combined organic extract waswashed with brine (50 mL), dried over anhydrous sodium sulfate, filteredand concentrated to provide 31 (2.57 crude, 97.5% yield) as white solid.This was used in the next step without further purification.

N-3'-(N-Oxysuccinimidylcarbonyl)Propyl2,3,4,5,6,7-Hexa-O-(3-Perfluorooctyl)Propionyl Glucoheptanamide 32

In a 100-mL round-bottom flask equipped with magnetic stirring bar andbent-tube adapter were placed compound 31 (2.57 g, 0.81 mmol), molecularsieves (100 mg), dimethylaminopyridine (122 mg, 1.0 mmol),N-hydroxysuccinimide (105 mg, 1.0 mmol), acetone (25 mL), and Freon®-113(25 mL) and cooled ˜5° C. To the above stirred mixture was addeddicyclohexylcarbodiimide (251 mg, 1.22 mmol) and the contents werefurther stirred for 18 h at room temperature. The mixture was filtered,concentrated and purified by "flash chromatography" to provide 32 (0.418g, 16% yield) as white solid (silica gel, ethyl acetate/Freon®-113 1:5).

¹ H NMR (CDCl₃ /Freon®-113 1:1) δ 1.747 (quint, 2H, H-2'), 2.08 (br,quart, 2H, H-3'), 2.616 (m, 28H, --COCH₂ CH₂ CF₂ --, --COCH₂ CH₂ CH₂CO--), 3.478 (m, 2H, H-1'), 4.167 (dd, 1H, H-7A, J_(H-7A),H-7B =12.7 Hz,J_(H-7A),H-6 =5.7 Hz), 4.342 (dd, 1H, H-7B, J_(H-7B),H-7A =12.4 Hz,J_(H-7B),H-6 =2.1 Hz), 5.208 (m, 1H, H-6), 5.248 (d, 1H, H-2,J_(H-2),H-3 =2.5 Hz), 5.528 (dd, 1H, H-3, J=H-3,H-2=2.5 Hz, J_(H-3),H-4=8.5 Hz), 5.981 (m, 2H, H-4 and H-5), 6.396 (br t, 1H, NH).

IR (KBr) 1740 (ester), 1815, 1786 (imide) 1680 (amide I), 1540 (amideII).

Monobenzyloxymethoxyhexaethyleneglycol 34

In a 500-mL bottom-round flask equipped with a magnetic stirring barwere placed 33 (16.4 mL, 65.5 mmol), anhydrous dichloromethane (150 mL),1,1,3,3-tetramethylurea (15.6 mL, 131 mmol) and stirred at -20° C. Tothe above mixture was added benzyl chloromethyl ether (9.1 mL, 65.5mmol). The contents were then stirred at -20° C. for 45 minutes and thenallowed to warm up to room temperature and further stirred for 18 h. Themixture was concentrated and chromatographed on silica gel (525 g,10:2:10 ethyl acetate, ethyl alcohol, hexane) to provide 34 (9.72 g, 37%yield).

¹ H NMR (300 MHz, CDCl₃) δ 2.75 (t, 1H, D₂ O exchange), 3.7 (m, 24H),4.61 (s, 2H, --OCH₂ Ph), 4.8 (s, 2H, --OCH₂ O--), 7.4 (m, 5H, aromatic).

(ω-Benzyloxymethoxy)Pentaethylenoxyethyl2,3,4,5,6-Tetra-O-Acetyl-β-D-Glucopyranoside 35

In a 250-mL round-bottom flask equipped with a magnetic stirring barwere placed 34 (4.02 g, 10 mmol), molecular sieves 4 Å (1 g), silvertriflate (2.56 g, 10 mmol), and anhydrous nitromethane (50 mL) andcooled to -20° C. To the above mixture was then added collidine (1.3 mL,10 mmol) and the mixture was further stirred at -20° C. for 15 minutesGlucosyl bromide 3 (4.92, 11 mmol) was then added and the contentsstirred at -20° C. for 18 h. The reaction mixture was diluted with ethylacetate and filtered through a Celite pad. The filtrate was washed with10% sodium thiosulfate and brine. The residue was chromatographed onsilica gel (200 g, 10:1:10 ethyl acetate, ethyl alcohol, hexane) toprovide 35 (724 mg, 10% yield).

¹ H NMR (300 MHz, CDCl₃) δ 2.007, 2.025, 2.037, 2.1 (4 s, 3H each,--OCOCH₃) 3.68 (m, 24H), 3.9 (m, 1H, H-5), 4.125 (dd, 1H, H-6A), 4.254(dd, 1H, H-6B), 4.61 (s, 2H, --OCH₂ Ph), 4.8 (s, 2H, --OCH₂ O--), 4.86(d, 1H, H-1), 5.0 (dd, 1H, H-2), 5.09 (dd, 1H, H-4), 5.21 (dd, 1H, H-3),7.4 (m, 5H, aromatic).

ω-(Benzyloxymethoxy)Pentaethylenoxyethyl β-Glucopyranoside 36

In a 100-mL round-bottom flask equipped with magnetic stirring bar wereplaced 35 (711 mg, 0.97 mmol), methanol (25 mL), and 0.5N sodiummethoxide (0.25 mmol), and stirred at 25° C. for 2 h, and thenneutralized with 500 mg of ion exchange resin AG-50W-X8 (Bio-Rad). Thecontents were stirred for 15 minutes, filtered and solvent removed toprovide 36 (525 mg, 96% yield).

¹ H NMR (300 MHz, D₂ O), δ 3.68 (m, 29H), 4.4 (d, 1H, H-1), 4.61 (s, 2H,CH₂ Ph), 4.8 (s, 2H, --OCH₂ O), 7.4 (s, 5H, aromatic).

(ω-Benzyloxymethoxy)Pentaethylenoxyethyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 37

In a 200-mL round-bottom flask equipped with magnetic stirring bar wereplaced 36 (485 mg, 0.86 mmol), molecular sieves 4 Å (100 mg),perfluorooctylpropionic acid (2.12 g, 4.3 mmol), dimethylaminopyridine(525 mg, 4.3 mmol), Freon®-113 (10 mL), stirred at 0° C. anddicyclohexylcarbodiimide (1.073 g, 5.2 mmol) in 10 mL ofdimethylformamide was added. The contents were stirred at roomtemperature for 18 h. The contents became very thick over the period. Itwas diluted with Freon®-113 (10 mL) and further stirred for 2 h,filtered, and residue chromatographed on silica gel (130 g, 1:1 ethylacetate, Freon®-113) to provide 37 (1.493 g, 71% yield).

¹ H NMR (300 MHz, Freon®-113+CDCl₃) δ 2.55 (bm, 8H, --CH₂ CF₂ --), 3.63(m, 24H, --OCH₂ CH₂ O--), 3.89 (m, 8H, --OCH₂ CH₂ CF₂), 3.95 (m, 1H,H-5), 4.2 (dd, 1H, H-6A), 4.46 (dd, 1H, H-6B), 4.62 (s, 2H, --OCH₂ O--),4.7 (d, 1H, H-1), 5.08 (dd, 1H, H-2), 5.2 (dd, 1H, H-4), 5.32 (dd, 1H,H-3), 7.3 (m, 5H, aromatic).

Pentaethylenoxyethyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 38

In a 250-mL round-bottom flask equipped with magnetic stirring bar wereplaced compound 37 (1.132 g, 0.46 mmol), palladium black (250 mg),Freon®-113 (90 mL) and 2,2,2-trifluoroethanol (18 mL) and stirred underhydrogen atmosphere for 18 h. The mixture was filtered through a Celitepad. The filtrate and washings were combined and the solvent was removedunder vacuum to provide 38 (966 mg, 93% yield). The compound wascharacterized by ¹³ C NMR.

EXAMPLE 1 Immobilization of Chymotrypsin Using4'-N-Oxysuccinimidylcarbonyl)Butyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside) 8

A one gram sample of fluorocarbon particles were washed with 10 mL ofHPLC grade acetone. The particles were then washed twice with 10 mLportions of 1:1 acetone-water. After the washes, the solution above thesettled particles were removed with a disposable pipet.Poly(fluoroalkyl) sugar reagent 8 (described on page 21) was dissolvedin acetone (2 mg/mL), then enough water was slowly added to give a 1:1acetone-water mixture. At this point the solution may become slightlycloudy. The reagent solution (10 mL) was added to 1 gram of the washedparticles and the suspension was stirred for 30 min to 1 h at roomtemperature to immobilize the reagent onto the particles. After thistime, the particles were washed twice with 10 mL portions of 1:1acetone-water, followed by four washes with water. During the waterwashes the particles were stirred gently with a small spatula to preventthe entrapment of air within the particles, which causes them to float.

To immobilize chymotrypsin, 10 mL of a solution of the enzyme (1 mg/mL)in 0.1M, pH 7.5 MOPS buffer was added to the activated particles, andthe mixture was stirred for 2 h at room temperature. Afterimmobilization, the particles were washed three times with MOPS buffer.Optionally, the particles were washed twice with 0.2% Zonyl® FSNfluorosurfactant in MOPS buffer for 15 min, followed by three morewashes with MOPS buffer.

The amount of chymotrypsin immobilized onto the particles was determinedby measuring the amount of protein remaining in the solution afterimmobilization and in the washes using a colorimetric protein assay.

The activity of chymotrypsin was determined using N-benzoyl-L-tyrosinep-nitroanilide (BTpNA) as substrate. The rate of production ofp-nitroaniline was measured at 385 nm. The molar absorption coefficientfor p-nitroaniline at this wavelength is 1.258×10⁴. The substratesolution contained nine volumes of 0.04M Tris buffer, pH 8.0, which was0.005M in calcium chloride and one volume of a solution of BTpNA inacetone (0.00178M). Aliquots of 2.9 mL of the substrate solution wereadded to 3 mL spectrophotometer cells, 0.1 mL of the diluted (1:10)stock enzyme solution was added, and the absorbance was measured at 2min intervals for 16 min.

To determine the enzyme activity immobilized on the particles, 50 uL ofthe settled suspension was added to 2.9 mL of the substrate solution.This mixture was incubated for 10 min at room temperature with vigorousstirring. The suspension was quickly transferred to a dry, preweighedcentrifuge tube and centrifuged for 1 min. The supernatant wastransferred to a spectrometric cell and the absorbance at 385 nm wasmeasured. The remaining particles were quantitatively transferred to thecentrifuge tube, washed several times with water, and dried in an ovenat 120° C. overnight. The dried particles were weighed the next day toobtain the weight of the particles used in the assay. The immobilizedenzyme activity per gram of particles and the retention of activity werecalculated.

The amount of chymotrypsin immobilized was 9.3 mg per gram of particles.The total enzymatic activity present on the particles was 1.2 IU pergram. The retention of enzymatic activity after immobilization was 33%of the original activity.

EXAMPLE 2 Immobilization of Chymotrypsin UsingN-3'-(N-Oxysuccinimidylcarbonyl)Propyl2,3,4,5,6-Penta-O-(3-Perfluorooctyl)Propionyl Gluconamide 25

The procedure described in Example 1 was used, except that thepoly(fluoroalkyl) sugar reagent 25 was substituent for Reagent 8. Theamount of chymotrypsin immobilized was 9.1 mg per gram of particles, andthe total activity was 1.2 IU per gram of particles. The retention ofenzymatic activity after immobilization was 31% of the originalactivity.

EXAMPLE 3 Immobilization of Chymotrypsin UsingN-3'-(N-Oxysuccinimidylcarbonyl)Propyl2,3,4,5,6,7-Hexa-O-(3-Perfluorooctyl)Propionyl Glyucoheptanamide 32

The procedure described in Example 1 was used, except that thepoly(fluoroalkyl) sugar reagent 32 was substituted for Reagent 8. Theamount of chymotrypsin immobilized was 8.6 mg per gram of particles, andthe total activity was 1.0 IU per gram of particles. The retention ofenzymatic activity after immobilization was 40% of the originalactivity.

EXAMPLE 4 Immobilization of Chymotrypsin Using4'-(N-Oxysuccinimidylcarbonyl)Butyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside) andNeutral Fluorosurfactant

Fluorocarbon particles (0.5 g) were activated with poly(fluoroalkyl)sugar reagent 8, as described in Example 1. To immobilize the enzyme,5.0 mL of a 1 mg/mL chymotrypsin solution in pH 7.5 MOPS buffer,containing 0.05% Zonyl FSN, was added to the particles. Chymotrypsinactivity was determined spectrophotometrically by measuring the increasein absorbance at 256 nm resulting from the hydrolysis ofbenzoyl-L-tyrosine-ethyl (ester (BTEE). The assay mixture contained 1.5mL of 0.08 Tris-HCl buffer, pH 7.8, with 0.1M calcium chloride, and 1.4mL of 1.07 mM BTEE in 50% methanol.

The addition of the fluorosurfactant to the enzyme solution did notaffect the amount of enzyme immobilized. However, the retention ofactivity increased to 50%.

EXAMPLE 5 Penicillin Amidase Immobilization Using4'-(N-Oxysuccinimidylcarbonyl)Butyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside)

One gram of fluorocarbon particles was activated with poly(fluoroalkyl)sugar reagent 8 as described in Example 1.

For penicillin amidase immobilization, the stock enzyme solution (950IU/mL, 81.5 mg protein/mL) was diluted ten-fold with 0.1M, pH 7.5 MOPSbuffer. Two milliliters of this enzyme solution was added to one gram ofactivated particles, and the suspension was stirred for 2 h at roomtemperature. The particles were washed three times using 0.02M, pH 8.0phosphate buffer. The amount of protein immobilized was determined fromthe depletion of enzyme activity from the original enzyme solution andthe activity present in the washes.

Penicillin amidase activity was determined using a method based on thetransformation of penicillin G into 6-aminopenicillinate andphenylacetic acid, catalyzed by penicillin amidase. The rate of thereaction was determined using a pH Stat to measure the rate ofconsumption of sodium hydroxide. The substrate sodium penicillin G wasdissolved in 0.02M, pH 8.0 phosphate buffer to give a 5% solution. Tenmilliliters of the substrate solution was cooled to the reaction vesselof the pH Stat, and 50 uL of enzyme solution was added. The rate ofaddition of 0.1M sodium hydroxide solution, required to maintain a pH of8.00, was recorded for 10 min. The specific activity of the enzyme wascalculated from the measured reaction rate. The immobilized enzymeactivity was determined in the same way as the soluble enzyme, using 100uL of the settled particles in 10.0 mL of the substrate solution. Theparticles were collected, washed with water, and dried after completionof the assay.

The amount of penicillin amidase immobilized was 5.6 mg per gram ofsupport, and the retention of activity was 51%. In control experimentsin which the fluorocarbon particles were not treated with the sugarreagent, 8.6 mg of penicillin amidase was adsorbed onto the fluorocarbonparticles, but no enzymatic activity was detectable.

EXAMPLE 6 Immobilization of Penicillin Amidase on Porous Silica Treatedwith Fluorosilanes A. Preparation of Porous Silica Based Support

Fifty grams of silica gel (Zorbax™ PSM 300) coated with zirconium oxideby the procedure as described in U.S. Pat. No. 4,600,646 to Stout,incorporated herein by reference, were heated and stirred in a mixtureof 350 mL of toluene and 40 mL of dimethylformamide (DMF) containing 57g of imidazole under a Dean-Start trap. A 50-mL fraction of thedistillate was removed and the residual silica mixture was allowed tocool to approximately 50° C. To attach a fluorosilane interlayer ontothe silica, the Dean-Stark trap was removed and 80 g ofheptadecafluoro-1,1,2,2-tetrahydrodecyldichloromethyl silane(HDF-silane) was added to the silica mixture. The silica-HDF-silanemixture was refluxed for two hours, cooled and filtered. TheHDF-silane-coated silica was then washed with 80% aqueoustetrahydrofuran (THF), resuspended in 350 mL of 80% aqueous THF andrefluxed for 5 minutes. The HDF-silane-coated silica was cooled to 50°C., filtered and twice washed with 200 mL of THF per wash. The coatedsilica was resuspended in 350 mL of 80% aqueous THF, refluxed for 10minutes, filtered, twice washed with 200 mL of THF per wash and twicewashed with 200 mL of Freon® TF per wash. The resulting coated silicawas dried first in air and finally in a vacuum oven at 110° C.

B. Immobilization on Porous Silica

One gram of porous silica (Zorbax™ PSM300), treated with fluorosilanewas activated with 100 mg of poly(fluoroalkyl) sugar reagent 8, asdescribed in Example 1. The amount of protein immobilized, determined asdescribed in Example 5, was 69 mg per gram of particles, and theretention of activity was 7%.

EXAMPLE 7 Immobilization of Protein A Using4'(N-Oxysuccinimidylcarbonyl)Butyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 8

One gram of fluorocarbon particles was washed twice with 10 mL portionsof HPLC-grade acetone. The particles were then washed twice with 10 mLportions of acetone/water (1:1 v/v). After the washes, the solutionabove the settled particles was removed with a disposable pipet.Poly(fluoroalkyl) sugar reagent 8 (3 or 4 mg) was dissolved in acetone(2 mL), and water (2 mL) was slowly added to give an acetone/water (1:1v/v) solution. At this point the solution may become slightly cloudy.The reagent mixture (4 mL) was added to 1 gram of the washed particlesand the suspension was stirred for 30-60 minutes at room temperature toimmobilize the reagent onto the particles. After this time, theparticles were washed three times with 10 mL portions of acetone/water(1:1 v/v), followed by three washes with 10 mL portions of cold 0.1Msodium phosphate buffer, pH 8.5. During the buffer wash the particleswere stirred gently to prevent the entrapment of air within theparticles, which causes them to float.

Protein A was immobilized by adding 1.4 or 1.8 mL of a cold protein Asolution (5 mg/mL) in 0.1M sodium phosphate buffer, pH 8.5, to theactivated particles, and stirring the mixture overnight at 4° C. Afterimmobilization, the particles were washed three times with 5 mL portionsof 0.1M sodium phosphate buffer, pH 8.5, followed by washing five timeswith 5 mL portions of 0.2% Zonyl® FSN fluorosurfactant in 0.1M sodiumphosphate buffer, pH 8.5. Finally, the particles were phosphate threetimes with 5 mL portions of 0.1M sodium phosphate buffer, pH 8.5.

The amount of protein A immobilized onto the particles were determinedby measuring the amount of protein remaining in the solution afterimmobilization and in the washes, using a colorimetric protein assay.

The activity of immobilized protein A was determined by measuring thebinding of human immunoglobulin (hIgG) to protein A at pH 8.0, followedby release of the hIgG at pH 3.0. The particles with immobilized proteinA were quantitatively packed into a small (1.25 mL) polypropylenechromatography column which was connected to a pump, set at a flow rateof 2.0 mL/min, and a UV detector, set at 280 nm, equipped with arecording integrator. The column was first equilibrated with 3 mL of0.1M sodium phosphate buffer, pH 8.0, and a baseline was established forthe integrator. Then 2.5 mL of a 10 mg/mL solution of hIgG in 0.1Msodium phosphate buffer, pH 8.0, was pumped onto the column followed by5.5 mL of the buffer. Finally 5 mL of 0.1M glycine-HCl buffer, pH 3.0,was pumped through the column to release the bound hIgG. The bindingcapacity of the immobilized protein A for hIgG was calculated bymultiplying the amount of hIgG loaded onto the column (25 mg) with thearea integrated for the released hIgG (as a fraction of the totalintegrated area). The volume of particles in the column was determinedand the hIgG binding capacity recalculated as mg/mL.

    ______________________________________                                        Results:                                                                             Poly(fluoro-       Immobilized                                                                            Binding                                    Ex-    alkyl) sugar                                                                             Protein protein  capacity for                               periment                                                                             reagent (mg)                                                                             A (mg)  A (mg)   hIgG (mg/mL)                               ______________________________________                                        1      3          7       3.0      9.0                                        2      3          9       3.5      12.0                                       3      4          7       5.5      9.2                                        4      4          9       6.9      11.3                                       ______________________________________                                    

EXAMPLE 8 Immobilization of Protein A UsingN-3'-(N-Oxysuccinimidylcarbonyl)Propyl2,3,4,6-Penta-O-(3-Perfluorooctyl)Propionyl Gluconamide 25

The procedure described in Example 7 was used, except that thepoly(fluoroalkyl) sugar reagent 25 was substituted for reagent 8.

    ______________________________________                                        Results:                                                                             Poly(fluoro-       Immobilized                                                                            Binding                                    Ex-    alkyl) sugar                                                                             Protein protein  capacity for                               periment                                                                             reagent (mg)                                                                             A (mg)  A (mg)   hIgG (mg/mL)                               ______________________________________                                        1      3          7       3.2      9.2                                        2      3          9       3.8      9.5                                        3      4          7       4.7      11.4                                       4      4          9       5.5      11.4                                       ______________________________________                                    

EXAMPLE 9 Immobilization of Protein A UsingN-3'-(N-Oxysuccinimidylcarbonyl)Propyl2,3,4,5,6,7-Hexa-O-(3-Perfluorooctyl)Propionylglucoheptanamide 32

The procedure described in Example 7 was used, except that thepoly(fluoroalkyl) sugar reagent 32 was substituted for reagent 8.

    ______________________________________                                        Results:                                                                             Poly(fluoro-       Immobilized                                                                            Binding                                    Ex-    alkyl) sugar                                                                             Protein protein  capacity for                               periment                                                                             reagent (mg)                                                                             A (mg)  A (mg)   hIgG (mg/mL)                               ______________________________________                                        1      3          7       3.0      6.7                                        2      3          9       4.7      10.7                                       3      4          7       4.0      5.2                                        4      4          9       3.0      5.0                                        ______________________________________                                    

EXAMPLE 10 Immobilization of Protein G Using4'(N-Oxysuccinimidylcarbonyl)Butyl2,3,4,6-Tetra-O-(3-Perfluorooctyl)Propionyl-β-D-Glucopyranoside 8

The procedure described in Example 7 was used, except that protein G wassubstituted for Protein A.

    ______________________________________                                        Results:                                                                             Poly(fluoro-       Immobilized                                                                            Binding                                    Ex-    alkyl) sugar                                                                             Protein protein  capacity for                               periment                                                                             reagent (mg)                                                                             G (mg)  G (mg)   hIgG (mg/mL)                               ______________________________________                                        1      4          4       2.7      7.9                                        2      4          9.5     3.8      11.0                                       ______________________________________                                    

As many differing embodiments of this invention may be made withoutdeparting from the spirit and scope thereof, it is to be understood thatthis invention is not limited to the specific embodiments exemplifiedexcept as defined by the appended claims.

We claim:
 1. A process for immobilizing an enzyme on a fluorocarbon surface comprising the steps of:(a) activating the fluorocarbon surface by contacting the surface with a reactive poly(fluoroalkyl) sugar reagent containing a polyhydroxy sugar to which are attached a plurality of fluoroalkyl anchor groups and a reactive group to produce an activated fluorocarbon surface; then (b) contacting the activated fluorocarbon surface with a solution of an enzyme, in the presence of a neutral fluoroalkyl-polyoxyethylene fluorosurfactant to covalently bond the enzyme to the reactive group of the poly(fluoroalkyl) sugar reagent to immobilize the enzyme on the fluorocarbon surface.
 2. The process of claim 1 wherein the sugar reagent contains a spacer.
 3. A process for immobilizing an enzyme on a fluorocarbon surface comprising the steps of:(a) activating the fluorocarbon surface by contacting the surface with a reactive poly(fluoroalkyl) sugar reagent containing a polyhydroxy sugar to which are attached a plurality of fluoroalkyl anchor groups, a spacer and a reactive group to produce an activated fluorocarbon surface; then (b) adding a fluoroalkyl-polyoxyethylene neutral fluorosurfactant; then (c) contacting the activated fluorocarbon surface with a solution of an enzyme to covalently bond the enzyme to the poly(fluoroalkyl) sugar reagent to immobilize the enzyme on the fluorocarbon surface.
 4. A process for immobilizing an enzyme on a fluorocarbon surface comprising the steps of:(a) activating the fluorocarbon surface by contacting the fluorocarbon surface with a reactive poly(fluoroalkyl) sugar reagent containing a polyhydroxy sugar to which are attached a plurality of fluoroalkyl anchor groups, a spacer and a reactive group, causing the fluorocarbon surface to absorb the reagent, in the presence of a fluoroalkyl-polyoxyethylene neutral fluorosurfactant to produce an activated fluorocarbon surface; then (b) contacting the activated fluorocarbon surface with a solution of an enzyme to attach the enzyme to the reactive group of the poly(fluoroalkyl) sugar reagent to immobilize the enzyme on the fluorocarbon surface. 